The long term objective of our research is the elucidation of the molecular mechanism of the acute and chronic effects of opiate narcotic analgesics. Our approach to this problem continues to be the study of the endogenous opioid system. Knowledge of the workings and functions of this system will have wide-ranging implications and, we feel, will lead towards an understanding of the actions of exogenous opiates. The focus of our research is an interdisciplinary exploration of the properties and functions of the major types or opioid receptors. Based on our success in purifying mu binding sites to homogeneity, we plan to apply our experience to the purification of other opioid receptor types, such as kappa and delta. The cloning and total sequencing of the mu receptor is in progress. We also plan to study the structure of the non-protein portion of receptor molecules, i.e., the carbohydrate portions of these glycoproteins and the nature of lipids essential for binding and function. The ultimate goal of all these studies is the knowledge of the complete structure of the binding portion of the opioid receptors. The interaction of the binding sires with G-proteins and second messenger systems as well as the possible role of receptor phosphorylation in receptor regulation will also be examined. The production of antibodies to purified receptors and to peptide fragments derived from them will be of enormous utility in many of our proposed studies and will be actively pursued. When the total amino acid sequence of one or more opioid receptor types is known, we plan to study receptor mRNA distribution and regulation using appropriate cDNA probes. Once we have established that the "wild type" receptor cDNA or gene can be expressed in a suitable mammalian cell line, we plan to use site-directed mutations and deletions to study the functional domains of the receptors. Purified receptors will be reconstituted into artificial and natural membranes and into cells normally devoid of opioid receptors. This has proved to be a powerful technique for the study of interactions of receptors with second messenger systems and ion channels. On the behavioral level we shall try determine the types of receptors and opioid peptides involved in analgesia, feeding and reward mechanisms. Behavioral as well as binding assays will be used to test new compounds synthesized by organic chemists collaborating with us.